This invention relates to a method of transferring a genetic substance directly into a plant's pollen after it is irradiated with ion beams of controlled energy to disrupt the shell of the pollen selectively so that the efficiency of transfer of a specific gene or DNA is enhanced. The method of the invention is broadly applicable to the breeding of plants.
Many techniques have been developed with a view to transferring an isolated useful gene into a plant species of interest. Such prior art techniques include: (1) a T-DNA method in which a specific gene is incorporated into a vector Ti plasmid of Agrobacterium, which is then infected to a plant to transfer the gene; (2) an electroporation method in which electric pulses are applied to a protoplast so that its cell membrane is disrupted temporarily and a gene is transferred into the cell; (3) a laser piercing method in which laser beams of a micron size is applied to a cell or tissue under examination with a microscope so that a hole is made momentarily and a gene is transferred into the cell or tissue through hole; and (4) a particle gun method in which fine metal particles of 1-3 .mu.m in diameter that are covered with a gene or DNA are injected into a cell or tissue with compressed air or the like so that the gene or DNA is transferred into the cell or tissue.
The T-DNA method requires two steps; in the first step, a vector incorporating a specific gene is transferred into an Agrobacterium by a freeze-thaw cycle, electroporation or some other suitable method; in the next step, a plant cell or slice is infected with the bacterium. In addition, due to the low infection of monocotyledons with Agrobacterium, the use of the T-DNA method is generally limited to dicotyledons. As a further problem, some dicotyledons are difficult to transform even if they are infected with Agrobacterium and this presents a need to set strict conditions. What is more, redifferentiated individuals are not easy to grow.
The electroporation method is applicable not only to dicotyledons but also to monocotyledons. However, a protoplast has to be prepared from isolated cells and the plant species that can be treated by this method are limited because the method is only applicable to those plants for which a cultivation system has been established to enable the growing or redifferentiated individuals from a proplast into which a gene has been transferred. Another problem with the electroporation method is the low efficiency of gene transfer since many cells die upon application of electric pulses.
The laser piercing method permits efficient gene transfer not only into cells but also to tissues such as hypototyl. However, due to the need of operation under examination with a microscope, it is difficult to treat a large volume of samples by this method. In addition, the method is not applicable to samples that cannot be manipulated under microscope and breeding of redifferentiated individuals is cumbersome.
The particle gun method is effective for plant species that have difficulty with culturing protoplasts and it permits direct gene transfer into viable cells having cell wall. The method has an additional advantage of enabling treatment of a large volume of samples. On the other hand, the method has to be operated under vacuum. In addition, many cells die under the impact of fine gold or tungsten particles and it is not easy to grow re-differentiated individuals. These facts contribute to the low efficiency of gene transfer that can be achieved by the particle gun method.
The number of plants which have an established cultivation system for regenerating plants from protoplasts and so forth is comparatively small. Regeneration may be possible with experimental plants it is often impossible with cultured plants. The prior art that can be used to create transgenic plant includes the T-DNA method, the laser piercing method and the particle gun method. In the T-DNA method, the plant species that can be transformed are limited. In this respect, physical techniques that involve less constraint are desirable. However, the laser piercing method which is a physical method of gene transfer is not applicable to samples that cannot be examined under microscope and it has an additional disadvantage of being incapable of treating a large volume of samples. In this respect, the particle gun method which is another physical technique is preferred.
Under the circumstances, the particle gun method is currently used widely for the purpose of transferring specific genes. In fact, however, many cells die under the great physical impact imposed by the particle gun method or on account of the vacuum under which the treatment is done. In addition, growing redifferentiated individuals is not easy. As a result, the efficiency of gene transfer is lowered. In order to solve these problems, a technique has to be developed that is capable of transferring a gene without causing significant damage to the cell nucleus.
If a specific gene can be transferred into a pollen grain, subsequent hybridization with the gene carrying pollen will permit gene transfer into fertile embryo, thus leading to the preparation of a pollen serving as a kind of vector. However, the shell of the pollen is strong both chemically and physically and cannot be removed by any of the prior art methods. Hence, it has been difficult to transfer a specific gene into the pollen.